Project Summary/Abstract: In the 130 years since Leber's Hereditary Optic Neuropathy (LHON) was described, no effective treatments for the disease have been found. Generations of young adults still lose sight, usually permanently. The demonstration of mitochondrial etiology has directed attention to finding treatments utilizing mitochondrial mechanisms, such as antioxidants and nuclear gene therapies to produce therapeutic proteins destined for the mitochondria. Nevertheless, no successful therapies have been developed. Gencia Corporation has developed a technology termed Protofection (Protein-Mediated Transfection) which is able to deliver full-length mitochondrial DNA (mtDNA) both in vitro and in vivo. Protofection is a DNA-binding, non-viral delivery vector consisting of an engineered recombinant protein that allows for transfection of mitochondria. After intraperitoneal injection, it delivers mtDNA to mitochondria in multiple tissues, including brain, muscle, and liver, and achieves robust mitochondrial expression of delivered DNA. The goal of this Phase I application is to show the feasibility of protofection as a treatment for LHON by supplementation of normal mitochondrial DNA to affected cells. The proposal will determine: (i) whether full- length mtDNA can be delivered to retinal ganglion cells (RGCs), the primary site of damage in LHON; (ii) the conditions necessary to achieve optimal delivery with minimal cytotoxicity; and (iii) the percentage of RGCs that can be transfected in vivo. Full-length mtDNA, engineered to have a new RFLP or to produce a marker protein (Green Fluorescent Protein, GFP), will be complexed with the protofection vector protein and delivered to the in vitro model of RGCs, the rat cell line RGC-5. The delivery of mtDNA to cells will be observed by following GFP fluorescence and by PCR for the introduced RFLP. TUNEL staining will be used as a measure of cellular toxicity. After establishing optimal dosage of mtDNA in vitro, analogous experiments will be performed in vivo through intravitreal injection of mtDNA complexed with the vector protein. The delivery of mtDNA will be tracked by GFP fluorescence in the retina and by RFLP PCR on the optic nerve. Successful achievement of the project goal will provide the basis for an extensive investigation into the efficacy and safety of this technology in a Phase II application. The goal of a Phase II application would be to conduct pre-clinical studies necessary for an IND application to the FDA and the Center for Biologics Evaluation and Research (CBER) for the treatment of LHON. Project Narrative: The aims carried out under this[unreadable] proposal will show feasibility for protofection as a treatment for Leber's hereditary optic neuropathy, a form of blindness caused by mutations in mtDNA and also provide a basis for treating other mitochondrial diseases. The potential impact on public health is significant. Although the incidence of most individual inherited mitochondrial diseases is low, taken as a group, they afflict an estimated 500,000 people in the U.S. alone. [unreadable] [unreadable] [unreadable]